The present invention relates to the development of a novel see-through medaka, a see-through medaka whose sex can be identified, and a see-through medaka in which specific organs produce luminescence.
Fifteen species belonging to genus Oryzias (Medaka) are known and are distributed in the Asian area from India to Japan. A species having its habitat in Japan is Oryzias latipes, which is also distributed in parts of China and Korea. With the exception of the Hokkaido area, it is distributed throughout Japan, and propagates during May to August in the natural world.
Common wild medaka has a dark body color and this is due to four types of pigment cells in the skin of medaka: iridophore, melanophore, xanthophore and leucophore. The iridophore is a silver pigment cell containing guanine granules. The iridophore is distributed prevalently in opercula, eyeballs, body wall and epidermis. In fish, it is distributed mainly in the ventral. The melanophore contains melanin granules and is involved in the dark body color of medaka. The xanthophore has pigment granules containing carotenoids and pteridines, and is involved in yellowish orange body color of medaka. The leucophore contains white granules compacted with pterine and uric acid, and is involved in white body color of medaka. Apart from the wild type which has a dark body color, there are known various mutants in relation to body color and morphogenesis. Among them, xe2x80x9chimedakaxe2x80x9d which has a yellowish orange body color is produced on a large scale as an aquarium fish and feed for large aquarium fishes.
Medaka is widely used as a simple and useful experimental animal due to a small body size of 2-4 cm, a short term for sexual maturation of 2 months, the fact that due to their wide distribution, the wild type population and closely-related species are easily obtainable and because its genome is ⅕ the size of mammals"", and sex determination is by X and Y chromosomes as in humans. The embryo of medaka is especially useful, since it is highly transparent with an easily observable internal structure. After hatching, however, since the epidermis and the peritoneum are covered with pigment cells, only the backbone can be seen through the body and the observation of the internal structure of the body from the outside becomes difficult.
Attempts have made recently at visualization of in vivo changes occuring in the internal structure by introducing a specific gene in Xenopus (amphibian) [Louie, A. Y. et al., In vivo visualization of gene expression using magnetic resonance imaging. Nature Biotechnology, 18, 321-325 (2000)]; and mice [Service, R. F., Scanners get a fix on lab animals. Science, 286, 2261-2263 (1999)], and tracing protein, a product of this gene, with MRI (magnetic resonance imaging), CT scanner (computed tomography scanner) or PET (position emission topography). These methods can, however, only provide insufficient images in spite of their requiring large and expensive equipment.
Further, there have been attempts at the lifelong tracing of the dynamics of certain organs by providing these organs with luminescence by means of GFP. Such studies are being performed with mouse tumor tissue, but observation can only be made at a maximum subcutaneous depth of 2 mm. [Yang, M. et al., Whole-body optical imaging of green fluorescent protein-expressing tumors and metastasis. PNAS, 97, 1206-1211 (2000)]. Expression of GFP fluorescence in transgenic medaka by introduction of a GFP gene fused with a medaka gene promoter into fertilized eggs, is known. [Hamada, K. et al., Usefulness of the medaka xcex2-actin promoter investigated using a mutant GFP reporter gene in transgenic medaka (Oryzias latipes). Mol. Marine Biol. Biotech., 7, 173-180 (1998)]. Tanaka, Minoru and Kinoshita, Masato, and Nagahama, Yoshitaka produced a transgenic medaka with green fluorescence in only germ cells as a result of introducing vasa-GFP gene into fertilized eggs of himedaka [in The 22nd Annual Meeting of the Molecular Biology Society of Japan, Program, Abstract, pp. 458 (1999), Tanaka, Minoru, Kinoshita, Masato and Nagahana, Yoshitaka, xe2x80x9cInbred medaka transgenics expressing GFP in germ cellsxe2x80x9d]. However, even in this transgenic medaka, fluorescence can only be observed up to the term immediately after hatching, and thereafter the body is covered with pigment cells and the observation becomes impossible.
For the above reasons, it is desirable that there be developed an experimental animal in which life-long observation of the internal structure of the body from the outside is possible. However, no such the experimental animal is known at present in vertebrates.
An object of the present invention is to provide a see-through medaka, in which the internal body structure thereof can be observed from the outside not only in the embryonic stage but also in the post-hatching stage. Another object of the present invention is to provide a see-through medaka, whose sex can be indentified. A further object of the present invention is to provide a see-through medaka, in which specific organs produce luminescence.
The inventors have studied extensively to solve the above problems, and have succeeded as a result of selective mating among mutant medaka, deficient in one or more types of pigment cells among the four types of pigment cells constituting dark color of medaka, to produce a medaka in which the internal body structure thereof can be observed from the outside not only in the embryonic stage but also in the post-hatching stage, thereby completed the present invention.
(i) The present invention relates to a see-through medaka wherein said medaka is deficient in iridophores, melanophores, xanthophores and leucophores.
(ii) Further, the present invention relates to the see-through medaka of (i) above wherein said medaka is produced by means of repeated selective mating between iridophore deficient mutant medaka strain gu, albino mutant medaka strain i-3 and leucophore deficient mutant medaka strain lf.
(iii) Further, the present invention relates to a see-through medaka wherein said medaka is produced by means of further selective mating between the see-through medaka in the (ii) above and iridophore deficient mutant medaka strain il-1.
(iv) The present invention relates further to a see-through medaka wherein said medaka is deficient in iridophores, melanophores and xanthophores, and wherein the sex of said medaka can be identified by the presence or absence of leucophores and/or a DNA marker.
(v) The present invention relates to the see-through medaka of (iv) above wherein said medaka is produced by means of repeated selective mating between iridophore deficient mutant medaka strain gu, albino mutant medaka strain i-3, leucophore deficient mutant medaka strain lf and medaka FLF strain which is deficient in leucophore in the female.
(vi) The present invention relates to a see-through medaka wherein said medaka is produced by means of further selective mating between the see-through medaka of (iii) above and the see-through medaka of (v) above.
(vii) Further, the present invention relates to the see-through medaka according to any one of (i) to (vi) above wherein a specific organ is allowed to produce luminescence by introducing a hybrid gene being a fusion of a promoter of a gene which expresses specifically in said organ with a coding region of a gene encoding a fluorescent protein.
(viii) Further, the present invention relates to the see-through medaka according to (vii) above wherein said gene encoding the fluorescent protein is a gene encoding a green fluorescent protein.
(ix) Further the present invention relates to the see-through medaka according to (vii) or (viii) above wherein said organ is a gonadal organ.
The present invention will be explained hereinbelow in detail,